Marisol S. Castaneto

High-resolution mass spectrometric metabolite profiling of a novel synthetic designer drug, N-(adamantan-1-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (STS-135), using cryopreserved human hepatocytes and assessment of metabolic stability with human liver microsomes

N-(Adamantan-1-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (STS-135) is a new synthetic cannabinoid in herbal incense products discussed on Internet drug user forums and identified in police seizures. To date, there are no STS-135 clinical or in vitro studies identifying STS-135 metabolites. However, characterizing STS-135 metabolism is critical because synthetic cannabinoid metabolites can possess pharmacological activity and parent compounds are rarely detectable in urine. To characterize the metabolite profile, human hepatocytes were incubated with 10 µmol/L STS-135 for up to 3 h. High-resolution mass spectrometry with software-assisted data mining identified 29 STS-135 metabolites. Less than 25% of STS-135 parent compound remained after 3 h incubation. Primary metabolites were generated by mono-, di- or trihydroxylation with and without ketone formation, dealkylation, and oxidative defluorination of N-fluoropentyl side chain or possible oxidation to carboxylic acid, some of them further glucuronidated. Hydroxylations occurred mainly on the aliphatic adamantane ring and less commonly on the N-pentyl side chain. At 1 h, phase I metabolites predominated, while at 3 h, phase II metabolites were present in higher amounts. The major metabolites were monohydroxy STS-135 (M25) and dihydroxy STS-135 (M21), both hydroxylated on the adamantane system. Moreover, metabolic stability of STS-135 (1 µmol/L) was assessed in human liver microsomes experiments. The in vitro half-life of STS-135 was 3.1 ± 0.2 min and intrinsic clearance (CLint ) was 208.8 mL · min(-1)  · kg(-1) . This is the first report characterizing STS-135 hepatic metabolic pathways. These data provide potential urinary targets to document STS-135 intake in clinical and forensic settings and potential candidates for pharmacological testing.

Urinary prevalence, metabolite detection rates, temporal patterns and evaluation of suitable LC-MS/MS targets to document synthetic cannabinoid intake in US military urine specimens.

Abstract Background: Identifying synthetic cannabinoid designer drug abuse challenges toxicologists and drug testing programs. The best analytical approach for reliably documenting intake of emerging synthetic cannabinoids is unknown. Primarily metabolites are found in urine, but optimal metabolite targets remain unknown, and definitive identification is complicated by converging metabolic pathways. Methods: We screened 20,017 US military urine specimens collected from service members worldwide for synthetic cannabinoids between July 2011 and June 2012. We confirmed 1432 presumptive positive and 1069 presumptive negative specimens by qualitative liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis including 29 biomarkers for JWH-018, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210, JWH-250, RCS-4, AM2201 and MAM2201. Specimen preparation included enzyme hydrolysis and acetonitrile precipitation prior to LC-MS/MS analysis. We evaluated individual synthetic cannabinoid metabolite detection rates, prevalence, temporal patterns and suitable targets for analytical procedures. Results: Prevalence was 1.4% with 290 confirmed positive specimens, 92% JWH-018, 54% AM2201 and 39% JWH-122 metabolites. JWH-073, JWH-210 and JWH-250 also were identified in 37%, 4% and 8% of specimens, respectively. The United States Army Criminal Investigation Command seizure pattern for synthetic cannabinoid compounds matched our urine specimen results over the time frame of the study. Apart from one exception (AM2201), no parent compounds were observed. Conclusions: Hydroxyalkyl metabolites accounted for most confirmed positive tests, and in many cases, two metabolites were identified, increasing confidence in the results, and improving detection rates. These data also emphasize the need for new designer drug metabolism studies to provide relevant targets for synthetic cannabinoid identification.

Metabolic characterization of AH‐7921, a synthetic opioid designer drug: in vitro metabolic stability assessment and metabolite identification, evaluation of in silico prediction, and in vivo confirmation

AH-7921 (3,4-dichloro-N-[(1-dimethylamino)cyclohexylmethyl]benzamide) is a new synthetic opioid and has led to multiple non-fatal and fatal intoxications. To comprehensively study AH-7921 metabolism, we assessed human liver microsome (HLM) metabolic stability, determined AH-7921s metabolic profile after human hepatocytes incubation, confirmed our findings in a urine case specimen, and compared results to in silico predictions. For metabolic stability, 1 µmol/L AH-7921 was incubated with HLM for up to 1 h; for metabolite profiling, 10 µmol/L was incubated with pooled human hepatocytes for up to 3 h. Hepatocyte samples were analyzed by liquid chromatography quadrupole/time-of-flight high-resolution mass spectrometry (MS). High-resolution full scan MS and information-dependent acquisition MS/MS data were analyzed with MetabolitePilot™ (SCIEX) using multiple data processing algorithms. The presence of AH-7921 and metabolites was confirmed in the urine case specimen. In silico prediction of metabolite structures was performed with MetaSite™ (Molecular Discovery). AH-7921 in vitro half-life was 13.5 ± 0.4 min. We identified 12 AH-7921 metabolites after hepatocyte incubation, predominantly generated by demethylation, less dominantly by hydroxylation, and combinations of different biotransformations. Eleven of 12 metabolites identified in hepatocytes were found in the urine case specimen. One metabolite, proposed to be di-demethylated, N-hydroxylated and glucuronidated, eluted after AH-7921 and was the most abundant metabolite in non-hydrolyzed urine. MetaSite™ correctly predicted the two most abundant metabolites and the majority of observed biotransformations. The two most dominant metabolites after hepatocyte incubation (also identified in the urine case specimen) were desmethyl and di-desmethyl AH-7921. Together with the glucuronidated metabolites, these are likely suitable analytical targets for documenting AH-7921 intake. Copyright

Performance characteristics of an ELISA screening assay for urinary synthetic cannabinoids

Synthetic cannabinoids are marketed as legal alternatives to cannabis, as routine urine cannabinoid immunoassays do not detect synthetic cannabinoids. Laboratories are challenged to identify these new designer drugs that are widely available and represent a major public health and safety problem. Immunoassay testing offers rapid separation of presumptive positive and negative specimens, prior to more costly and time-consuming chromatographic confirmation. The Neogen SPICE ELISA kit targets JWH-018 N-pentanoic acid as a marker for urinary synthetic cannabinoids. Assay performance was evaluated by analyzing 2469 authentic urine samples with the Neogen immunoassay and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two immunoassay cut-off concentrations, 5 and 10 µg/L, classified samples as presumptive positive or negative, followed by qualitative LC-MS/MS confirmation for 29 synthetic cannabinoids markers with limits of detection of 0.5-10 µg/L to determine the assays sensitivity, specificity and efficacy. Challenges at ±25% of each cut-off also were investigated to determine performance around the cut-off and intra- and inter-plate imprecision. The immunoassay was linear from 1 to 250 µg/L (r(2)  = 0.992) with intra- and inter-plate imprecision of ≤5.3% and <9%, respectively. Sensitivity, specificity, and efficiency results with the 5 µg/L cut-off were 79.9%, 99.7%, and 97.4% and with the 10 µg/L cut-off 69.3%, 99.8%, and 96.3%, respectively. Cross-reactivity was shown for 18 of 73 synthetic cannabinoids markers evaluated. Good sensitivity, specificity, and efficiency, lack of sample preparation requirements, and rapid semi-automation documented that the Neogen SPICE ELISA kit is a viable method for screening synthetic cannabinoids in urine targeting JWH-018 N-pentanoic acid.

Method validation of the biochip array technology for synthetic cannabinoids detection in urine

BACKGROUND Synthetic cannabinoids (SC) are widely-abused cannabimimetic drugs that do not screen positive in traditional cannabinoids immunoassays, making detection difficult. METHODS AND RESULTS The first commercially-available immunoassay for urinary SC was validated. Limits of detection (5-20 µg/L), imprecision (<13.1% intra-, <37.7% inter-assay), and cross-reactivity profiles of 22 SC and 37 metabolites were obtained. A large negative bias (-80.8 to -28.0%) was observed. Sensitivity (98.3%), specificity (48.1%) and efficiency (53.9%) were determined from screening 20,017 urine specimens and confirming 1432 presumptive positive and 1069 selected negative specimens by LC-MS/MS. Cutoff optimization improved performance to 87.6% sensitivity, 85.2% specificity, and 85.4% efficiency. CONCLUSION This high-throughput urine SC assay has good sensitivity and improved specificity and efficiency at modified cutoff concentrations.

Identifying methamphetamine exposure in children.

Objective: Methamphetamine (MAMP) use, distribution, and manufacture remain a serious public health and safety problem in the United States, and children environmentally exposed to MAMP face a myriad of developmental, social, and health risks, including severe abuse and neglect necessitating child protection involvement. It is recommended that drug-endangered children receive medical evaluation and care with documentation of overall physical and mental conditions and have urine drug testing. The primary aim of this study was to determine the best biological matrix to detect MAMP, amphetamine (AMP), methylenedioxymethamphetamine (MDMA), methylenedioxyamphetamine (MDA), and 3,4-methylenedioxyethylamphetamine (MDEA) in environmentally exposed children. Methods: Ninety-one children, environmentally exposed to household MAMP intake, were medically evaluated at the Child and Adolescent Abuse Resource and Evaluation Diagnostic and Treatment Center at the University of California, Davis Children’s Hospital. MAMP, AMP, MDMA, MDA, and MDEA were quantified in urine and oral fluid (OF) by gas chromatography mass spectrometry and in hair by liquid chromatography tandem mass spectrometry. Results: Overall drug detection rates in OF, urine, and hair were 6.9%, 22.1%, and 77.8%, respectively. Seventy children (79%) tested positive for 1 or more drugs in 1 or more matrices. MAMP was the primary analyte detected in all 3 biological matrices. All positive OF (n = 5), and 18 of 19 positive urine specimens also had a positive hair test. Conclusions: Hair analysis offered a more sensitive tool for identifying MAMP, AMP, and MDMA environmental exposure in children than urine or OF testing. A negative urine or hair test does not exclude the possibility of drug exposure, but hair testing provided the greatest sensitivity for identifying drug-exposed children.

Urine Mescaline Screening With a Biochip Array Immunoassay and Quantification by Gas Chromatography-Mass Spectrometry.

Abstract: Mescaline, the primary psychoactive chemical in peyote cactus, has been consumed for thousands of years in ancient religious ceremonies. The US military wanted to determine if mescaline intake was a problem for personnel readiness. Twenty thousand seventeen urine specimens negative for cannabinoids, cocaine, opiates, and amphetamines were tested for mescaline with the Randox Drugs of Abuse V (DOA-V) biochip array immunoassay at the manufacturers recommended cutoff of 6 mcg/L. A sensitive and specific method for mescaline quantification in urine was developed and fully validated. Extracted analytes were derivatized with pentafluoropropionic anhydride and pentafluoropropanol and quantified by gas chromatography–mass spectrometry (GC/MS) with electron impact ionization. Standard curves, using linear least squares regression with 1/x2 weighting, were linear from 1 to 250 mcg/L with coefficients of determination >0.994. Intra- and inter-assay imprecision was <4.4 coefficient of variation (%CV), with accuracies >90.4%. Mean extraction efficiencies were >92.0% across the linear range. This fully validated method was applied for the confirmation of urinary mescaline in 526 presumptive-positive specimens and 198 randomly selected presumptive-negative specimens at the manufacturers 6 mcg/L cutoff. No specimen confirmed positive at the GC/MS limit of quantification of 1 mcg/L. Results indicated that during this time frame, there was insufficient mescaline drug use in the military to warrant routine screening in the drug testing program. However, mescaline stability, although assessed, could have contributed to lower prevalence. We also present a validated GC/MS method for mescaline quantification in urine for reliable confirmation of suspected mescaline intake.